Understanding the AIE phenomenon of nonconjugated rhodamine derivatives via aggregation-induced molecular conformation change

The bottom-up molecular science research paradigm has greatly propelled the advancement of materials science. However, some organic molecules can exhibit markedly different properties upon aggregation. Understanding the emergence of these properties and structure-property relationship has become a new research hotspot. In this work, by taking the unique closed-form rhodamines-based aggregation-induced emission (AIE) system as model compounds, we investigated their luminescent properties and the underlying mechanism deeply from a top-down viewpoint. Interestingly, the closed-form rhodamine-based AIE system did not display the expected emission behavior under high-viscosity or low-temperature conditions. Alternatively, we finally found that the molecular conformation change upon aggregation induced intramolecular charge transfer emission and played a significant role for the AIE phenomenon of these closed-form rhodamine derivatives. The application of these closed-form rhodamine-based AIE probe in food spoilage detection was also explored.

2. The molecules showed high QY, please provide the absorption coefficient of the molecules.
On page 8 of the revised manuscript, "As shown in Figure S14, the four compounds displayed similar characteristic absorption spectra with several sharp bands between 240 nm to 330 nm in THF solutions, which could be attributed to their intramolecular n-π* transition and π-π* transition.The molar absorption coefficient of BISX, ISX, MISX and MTSX is measured as 7446 M-1•cm-1, 7146 M-1•cm-1, 7700 M-1•cm-1, and 7566 M-1•cm-1, respectively." On page 14 of the revised SI,  3. In page 17 line 294, the authors should revise "change of molecular structure" into "change of molecular conformation", since there was no change in the molecular composition.In page 17 line 295, "structure" should also be replaced by "conformation".
Response: Thank you so much for the valuable comment.We have made corrections in the revised manuscript.
Response: Thank you so much for pointing out the typos.We have made corrections in the revised manuscript, and carefully went through the manuscript.
On page 3 of the revised manuscript, "In this work, by taking the unique closed-form rhodamines-based aggregation-induced emission (AIE) system as model compounds, ......"

5.
In page 4 line 70, the phrase "in the fluorescence area" should be changed into "in the field of fluorescence".
Response: Thank you so much for the valuable comment.According to the suggestions of reviewer 2, we have re-composed the introduction part and used the expression you suggested "in the field of XX".
On page 4 of the revised manuscript, "Furthermore, in the field of biology, the folded 3D structure of……" 6.In page 22 line 400, reference 1 does not contain page number; In page 23 line 424, reference 12 abbreviation of journal should be spaced.
Response: Thank you so much for the valuable comment.We have corrected ref 1 in the revised manuscript.And according to the suggestions of reviewer 2, we have re-composed the introduction part and deleted some references (including ref 12).We have carefully gone through the reference part.
On page 22 of the revised manuscript, "1.Ye, S. et al.Machine learning-assisted exploration of a versatile polymer platform with charge transfer-dependent full-color emission.Chem 9, 924-947 (2023)." 7. The formats of λex should be unified in all figure captions.
Response: Thank you so much for the valuable comment.We have unified them in the revised manuscript.
On page 14 of the revised manuscript, Response: Thank you so much for the valuable comment.We have remeasured the lifetime of each compound.
On page 15-16 of the revised SI,    Reviewer 2: This manuscript investigates aggregation induced emission (AIE) in rhodamine B derivatives.The authors describe their findings in term of "symmetry breaking boosted intramolecular charge transfer", and claim that their work "offers a deeper and more general understanding on the variations between single molecules and aggregates".While I fully appreciate the importance of AIE, the role of symmetry breaking, as described by the authors, is not clear and I find the proposed idea not helpful to understand the effect of aggregation.I appreciate that the explanation of AIE in this particular system is not straightforward, but the explanation provided by the authors is not satisfactory and the manuscript is not ready for publication in a high profile journal.I recommend rejection.See detailed comments.
Response: We thank you very much for your precious time and comments to review our manuscript and are grateful for your appreciation on the importance of AIE.We admit that our previous explanation of AIE in this particular system is not straightforward for readers to understand.According to your professional and constructive comments, we had a careful discussion and made extra experiments.We reproposed the working mechanism of these AIEgens as molecular conformation change induced by aggregation, which boosted the intramolecular CT transition in the aggregate state.We also re-structured the introduction part, and updated the title and TOC graph.
1.The meaning of the term 'symmetry' as used by the authors is not clear because it is ambiguous, and this makes understanding the general idea of the paper difficult.In the context of Landau theory, the 'symmetry breaking' that occurs during the phase transition probably refers to the transition from an isotropic to an anisotropic environment.However, the term symmetry has other uses that are not consistent with the one in Landau theory, eg crystalline systems are more symmetric than solutions considering that they have crystallographic point groups.This makes the term ambiguous and not very useful.In addition, whatever name one might give to it, the phenomenon described by Landau theory occurs for any system undergoing aggregation, ie it might be invoked to explain AIE in any AIEgen and not just the current one.It is not clear what particular role it plays here.
Going deeper into the specific explanation offered by the authors, their arguments refer to the structural changes that the molecule undergoes in the ground and excited states, both in solution and in the crystal.
In fact, this mechanistic explanation lies perfectly in line with the RIM model, the difference with other systems being that here the structural differences between solution and crystal are quite small.These small differences might explain, for instance, the lack of temperature and viscosity effects.Therefore I do not see how the ambiguous 'symmetry breaking' idea contributes to understanding AIE in this system.
Response: Thank you so much for your valuable comment.After careful consideration, we agree with you that the terms used "symmetry" and "symmetry breaking" are not clear.The "symmetry breaking" in our system refers to the disorder and order extent of the system in solution and crystal.We think it could help us to get a general understanding of the property difference between different states from the level of philosophy.However, indeed, it could not provide detailed information for the mechanism of the given specific AIE system we presented.Based on these considerations, we reorganized the introduction part with more focus on the science part but less attention on the philosophy part.According to the experimental results, we concluded that the aggregation could cause a conformation change, which could further induce an intramolecular charge transfer emission.Thus, we have revised the introduction part and the mechanism discussion.We also updated the title and TOC Graph to keep consistent.rhodamines-based aggregation-induced emission (AIE) system as model compounds, we investigated their luminescent properties and the underlying mechanism deeply from a top-down viewpoint.Interestingly, the closed-form rhodamine-based AIE system did not display the expected emission behavior under high-viscosity or low-temperature conditions.Alternatively, we finally found that the molecular conformation change upon aggregation induced intramolecular charge transfer emission and played a significant role for the AIE phenomenon of these closedform rhodamine derivatives.The application of these closed-form rhodamine-based AIE probe in food spoilage detection was also explored.

Introduction
Unveiling the structure-property relationship of organic functional materials and proposing effective materials design principle are among the most fascinating tasks for material scientists. 1,2,3Current molecular design of functional materials mainly follows a bottom-up molecular science research paradigm, which emphasizes the role of molecular structure in determining the properties of materials. 4,5,6Under the guidance of this molecular structure dominated research paradigm, various molecular materials with new structures have been designed through an experience-based subconsciousness.Taking organic luminescent materials as an example, the introduction of conjugated rigid building blocks and the expansion of the πconjugation are the commonly used strategies to develop new luminescent materials since they are generally thought necessary for the formation of chromophore and tuning the band gap. 7,8,9nother example is the preparation of chiral materials or drugs, which usually needs the incorporation of chiral factors or asymmetry catalysis to induce the generation of a chiral center. 10t is no doubt that the molecular structure dominated research paradigm has made significant contributions to the advancement of modern chemistry and materials. 11However, this molecular structure oriented materials development approach also suffers some emerging limitations with the expansion of the materials scope.There are more and more cases indicated that the molecular structure alone cannot fully determine the materials properties while their aggregation modes greatly influence their macroscopic performance. 12,13For instance, Yuan and coworkers have discovered that the chromophore-free saccharide molecules without even one phenyl ring could exhibit strong luminescence upon aggregation, and polypeptide and some other nonconjugated polymers exhibited similar phenomenon. 14,15Tang and coworkers discovered another type interesting luminescence phenomenon that some propeller-shaped molecules exhibit no emission in their isolated state but show bright emission in aggregation state, which they has coined as aggregation-induced emission (AIE). 16,17Li and Zhang et al. reported that the achiral molecules like hexaphenylsilole and tetraphenylethene (TPE) derivatives with non-chiral centers could generate circular dichroism signals and even circularly polarized light emission in aggregates while their single molecular state didn't show any chiral signals. 18,19,20Furthermore, in the field of biology, the folded 3D structure of polypeptide and its wrongly folded structure-resulted aggregates could also exhibit vastly different biological activities. 21,22These inconsistences between molecular properties and aggregate properties can possibly be ascribed to the complex changes occurring at both molecular level and aggregate level during the aggregation process, which unfortunately has long been ignored as the role of molecular structure is overpriced.Therefore, it is highly desirable to revisit the structure-property relationship from a top-down perspective with more attention focus on the influence brought by aggregation to the molecules.
By shifting the research interest from single molecule to aggregates at condensed state, scientists have unveiled that lots of factors induced by the multibody interactions play an important role in determining the materials performance. 23,24,25For example, An at al. reported that Haggregation in molecular packing could stabilize triplet excitons to realize ultralong organic phosphorescence. 26Li et al. established the one-to-one correspondence between the determinate interactions and excited triplet states, which guided the design of organic phosphorescence materials. 27Barrett at al. reported that co-crystallization of two or more molecules can exhibit properties that do not belong to any individual molecules. 28,29,30These works also highlighted how did the crystallinity, aggregate morphology, assembly behavior that occurred among multibodies affect the materials performance. 31,32In addition to multibody interactions, whether the aggregation will cause the change of the characteristic of molecule itself, like the molecular conformation or molecular electronic structure, leading to emerging of new macroscopic materials performance is also an interesting topic that is worth to investigated.However, investigation from this top-down viewpoint has seldomly been considered and there are very few related studies.Thus, it is crucial to investigate how the spatial confinement resulting from aggregation and changes in the surrounding molecular environment affect molecular characteristic like the molecular conformation.In other words, further research is needed to understand the differences in molecular conformation between the single molecular state and the aggregate state.This investigation will provide valuable insights into the influence of aggregation on materials properties and enable the development of strategies to optimize their performance.""Through a top-down viewpoint, we finally found that the molecular conformation change upon aggregation induced intramolecular charge transfer emission and played a significant role in the AIE phenomenon of these closed-form rhodamine derivatives." On page 17 of the revised manuscript, "Compared to the free molecules with high rotational and translational flexibility in the solution state, molecules within the confined aggregate state were interfered by various interactions (C-H•••π, π-π, hydrogen bonding), leading to conformation change.Thus, the conformation change in the aggregate state further boosted the transition allowed CT D-A emission, affording the AIE feature of these nonconjugated molecules." On page 19 of the revised manuscript, "Combined with analysis from single-crystal structure, optical properties investigation, and theoretical calculations, we proposed that molecular conformation change within confined microenvironment in aggregates boosted the intramolecular CT transition, which caused their luminescence variations between the solution and aggregation state." 2. The mechanistic hypotheses need more support from the calculations.In figure 5a, the authors invoke two excited states, LE and CT.What is the evidence for that?The orbitals in Figure S48 suggest that S1 is a CT state, why do the authors claim that emission in solution occurs from the LE state?The insight one can obtain from the frontier molecular orbitals provided in the manuscript is limited, a more complete study is required to obtain more insights into the mechanism.
Response: Thank you so much for your valuable comment.After careful thinking on your constructive comment, we found that we confused and misunderstood the concepts of LE and CT.The LE in our system was relative to the whole molecule.It referred to the electron transition occurred only at the upper part of the molecule, and CT referred to the electron transition occurred between the upper (Acceptor) and lower (Donor) parts of the molecule.Thus, to make it clearly and avoid confusion, we deleted the descriptions of "LE", and revised the two excited states as "change of electron clouds mainly took place on A moiety (CT A )", and "charge transfer from D to A (CT D-A )".CT A caused the short-wavelength emission at low temperatures, while CT D-A caused the long-wavelength AIE emission.Taking BISX as an example, we tested its temperature-dependent PL spectra in THF (Figure 2b and c).At 25 °C, the compound showed a hardly noticeable emission around 540 nm.However, upon cooling to -196 °C, a significant enhancement of fluorescence peak at 410-420 nm was observed, which corresponded to the fluorescence peaks of BIPM (analogue of the upper part of BISX).Therefore, in the solution state, BISX molecules gave almost no fluorescence due to their ability to move freely at 25 °C.However, as the temperature decreased, the molecular motion was suppressed.And since the molecules were in a dispersed state with minimal intermolecular interactions, only the upper part of BISX gave the fluorescence (CT A ) due to RIM.As shown in Figure S49, we further conducted calculations on BISX and its upper part, isoquinolinone.We found that BISX exhibited CT state only in its upper part, from HOMO-3 to LUMO, which is very similar to the electron cloud distribution of isoquinolinone from S₁ to S₀.Therefore, we assumed that in the solution state, the CT D-A of BISX is forbidden due to its perpendicular D-A structure, which caused electron clouds changes mainly took place on A moiety (CT A ).While in the aggregate state, aggregation helps break the transition forbidden in the solution state, facilitating the allowed CT D-A transition (Figure 5b and S50).
On page 15 of the revised manuscript, "…Further calculations proved that change of electron clouds mainly took place on A moiety of BISX (CT A ), almost the same with that of isolated isoquinolinone molecule from S 1 → S 0 (Figure S49).Thus, in the solution state, the system was almost non-emissive, most excitons will come to the ground state from the CT A state rather than charge transfer from D to A (CT D-A ) excited state, making CT D-A become a dark state (Figure 5a).This diagram also well accounted for the emission behavior of BISX in the previous cooling experiments (Figure 2b-c).At room temperature, due to the flexible molecular motion, the absorbed energy was mainly dissipated via the nonradiative decay pathway, affording negligible CT A emission.And the weak CT D-A emission which is responsible for the AIE peak was ascribed to a dark state.Although reducing temperature to restrict the molecular motion benefits enhancing the CT A emission at 430 nm, it affects scarcely the forbidden CT D-A emission of the perpendicular D-A structure." On page 16 of the revised manuscript, "…The improved HOMO and LUMO overlap benefits effective CT D-A transition to undergo radiative decay.Therefore, the aggregation not only helps break the transition forbidden in the solution state but facilitates the allowed CT D-A transition in aggregate state (Figure 5b and S50).Based on the single-crystal structure, QM/MM calculations were further performed by the ONIOM model in the Gaussian 16 package (Figure S51).As shown in Figure 5c, the variation of the dihedral angle between the excited state and ground state was merely 2.45°, leading to more accessible structural relaxation to the geometry of allowed CT D-A transition.Thus, in the aggregate state, BISX displayed CT D-A emission at 540 nm.Compared to the free molecules with high rotational and translational flexibility in the solution state, molecules within the confined aggregate state were interfered by various interactions (C-H•••π, π-π, hydrogen bonding), leading to conformation change.Thus, the conformation change in the aggregate state further boosted the transition allowed CT D-A emission, affording the AIE feature of these nonconjugated molecules." 3. Regarding the measured excited-state life times in Figures S17-S20, the authors argue that the life times in the crystal are longer by one order of magnitude, but the curves shown for the crystal have a very short, probably sub-ns component that seems to have been neglected (eg in figure 17b, the intensity has a sharp initial drop from approx.10^4 to 5*10^2).Has this initial drop been included in the fitting?Can it be explained?Please also explain what is exactly measured in these figures (eg transient absorption, fluorescence, etc).
Response: Thank you so much for your valuable comment.After careful checking, we found that sub-ns component was neglected, and the crystal lifetimes were not fully decayed in our previous measurement.Thus, we re-measured the time-resolved fluorescence decay curves of the four compounds.
On page 15-16 of the revised SI,    4. Turning to the application, the authors state in line 314 that in acid media, the sp2 hybridized nitrogen is more likely to be protonated than the sp3 one.However, the diethylamine substituents of the dibenzopyran moiety are expected to be even more basic than the sp2 nitrogen.How does this affect the proposed sensor mechanism?
Response: Thank you so much for your valuable comment.After carefully checking the electrostatic potential of BISX (Figure S52), we found that the electronegativity for the sp2 hybridized nitrogen is stronger than the nitrogen from the diethylamine substituents, so the sp2 hybridized nitrogen should more easily to be protonated.For further prove it, we calculated the frontier molecular orbitals of protonated compounds of BISX based on their optimized ground-state geometries for protonated sp2 hybridized nitrogen (Figure S53 a) and the protonated nitrogen from diethylamine substituents (Figure S53 b).The unstable isomer of the protonated structure displayed a higher electronic energy of 17.7 kcal/mol than that of the stable one.Thus, we assumed that the sp2 hybridized nitrogen should be first protonated.
On page 18 of the revised manuscript, "The calculated electron density maps based on electrostatic potential further verified our hypothesis, by giving the result that sp 2 N is more electron-rich than sp 3 N and the diethylamine substituents (Figure S52c).To further prove it, we calculated the frontier molecular orbitals of protonated compounds of BISX based on their optimized ground-state geometries (Figure S53).It was found that the protonated structure with a protonated diethylamine substituent is unstable, exhibiting a higher electronic energy of 17.7 kcal/mol compared to the protonated structure with a protonated sp 2 N." On page 34 of the revised SI,

Figure S53
Frontier molecular orbitals of protonated compounds of BISX based on their optimized ground-stategeometries for protonated sp2 hybridized nitrogen (a) and the protonated nitrogen from diethylamine substituents (b).

Figure 6 .
Figure 6.(a) Normalized PL spectra (a) and fluorescence images (b) of BISX, ISX, MISX and MTSX in the aggregate state.ex = 365 nm.(c) Maximal PL intensity of BISX treated with trifluoroacetic acid (TFA) and triethylamine (TEA).Inset: fluorescence images taken under 365 nm UV irradiation, respectively.(d) Spoilage detection of clams in sealed packages for 24 h at room temperature using BISX.Photographs taken under daylight (upper) and 365 nm UV irradiation (bottom).

Figure 4 .
PL spectra of BISX, ISX, MISX and MTSX (a-d) in PMMA film with different weight ratios (m/m), respectively.Inset: fluorescence images of the corresponding compounds in PMMA (5%). ex = 365 nm."On page 19 of the revised manuscript, "Figure 6.(a) Normalized PL spectra ... ex = 365 nm...." 8. Lifetime values of the AIE dyes should be provided to better understand the origin of the emission peaks.

Figure
Figure S18 (a) Time-resolved fluorescence decay curves of ISX in THF at 550 nm, c = 1×10 -5 M. (b) Time-resolved fluorescence decay curves of ISX crystal at 515 nm.
Title: Understanding the AIE phenomenon of nonconjugated rhodamine derivatives via aggregation-induced molecular conformation change TOC graph: On page 3-6 of the revised manuscript, "Abstract The bottom-up molecular science research paradigm has greatly propelled the advancement of materials science.However, some organic molecules can exhibit markedly different properties upon aggregation.Understanding the emergence of these properties and structure-property relationship has become a new research hotspot.In this work, by taking the unique closed-form

Figure S49 .
Figure S49.The ground state distribution of frontier molecular orbitals of BISX (top), and isoquinolinone (bottom) molecules in THF solutions.

Figure
Figure S18 (a) Time-resolved fluorescence decay curves of ISX in THF at 550 nm, c = 1×10 -5 M. (b) Time-resolved fluorescence decay curves of ISX crystal at 515 nm.